TMAO enhances TNF-α mediated fibrosis and release of inflammatory mediators from renal fibroblasts

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.


Proliferation assay
Renal fibroblasts were stimulated with TMAO (300 µM), TNF-α (1, 10 or 50 ng/ml) or the combination of both for 48 h and incubated at 37ºC with 5% CO 2 .Renal fibroblasts were pre-stimulated with TMAO for 2 h prior to TNF-α stimulation during the combination treatments.After stimulation, the renal fibroblasts were washed once with PBS.0.1% Crystal violet (diluted in 20% methanol, Sigma-Aldrich) was then added to the fibroblasts for 10 min at room temperature and the cells were then washed twice with tap water.The renal fibroblasts were then destained with 1% sodium dodecyl sulfate on a shaker at 500 rpm for 5 min and evaluated at 570 nm using the Cytation 3 plate reader.

Total collagen production
Renal fibroblasts were stimulated with TMAO (300 µM), TNF-α (1, 10 or 50 ng/ml) or the combination of both in the presence of 50 µg/ml sodium ascorbic acid (Thermo fisher Scientific) for 96 h incubated at 37ºC with 5% CO 2 .Renal fibroblasts were pre-stimulated with TMAO for 2 h prior to TNF-α stimulation during the combination treatments.After stimulation, total collagen production was evaluated using Sirius red staining (Thermo fisher Scientific).The renal fibroblasts were incubated with 1 mg/ml Sirius red (diluted in picric acid) for 30 min at room temperature.The fibroblasts were then washed with PBS and destained with NaOH 0.1 M on a shaker at 700 rpm for 15 min.The destaining solutions were transferred to a new 96-well plate and evaluated at 540 nm using the Cytation 3 plate reader.

Targeted protein analysis
The renal fibroblasts were stimulated with TMAO (300 µM), TNF-α (1, 10 or 50 ng/ml) or the combination of both for 24 h and incubated at 37ºC with 5% CO 2 .Renal fibroblasts were pre-stimulated with TMAO for 2 h prior

TMAO and TNF-α promote renal fibroblast proliferation via Akt/mTOR and ERK pathways
Next, we wanted to elucidate whether the combination of TMAO and TNF-α could increase renal fibroblast proliferation.We found that TMAO 300 μM and TNF-α 1 ng/ml alone or in combination, significantly increased renal fibroblast proliferation compared to unstimulated cells after 48 h (Fig. 2A).Notably, the combination of TNF-α 1 ng/ml and TMAO caused a significant increased proliferation compared to TNF-α 1 ng/ml alone (Fig. 2A).However, this increase was not synergistic.Next, we investigated which signaling pathways mediate the proliferative effect of TMAO and TNF-α on renal fibroblasts.We found that inhibition of Akt (MK-2206), mTOR (Ridaforolimus), ERK (PD98059), but not PI3K (Wortmannin), resulted in reduced cell proliferation after stimulation with TMAO and TNF-α alone or in combination compared to DMSO treated cells after 48 h (Fig. 2B).Taken together, our results show that TMAO and TNF-α mediate their proliferative effect on renal fibroblast using the Akt/mTOR and ERK signaling pathways.

TMAO and TNF-α synergistically increases total collagen production via Akt/mTOR and ERK pathways
We continued to investigate whether TMAO could enhance TNF-α mediated total collagen production from renal fibroblasts.We found that TMAO 300 μM and TNF-α alone or in combination increased total collagen production compared to unstimulated cells (Fig. 3A).We also found that the combination treatments of TNF-α 1 ng/ml and TMAO significantly induced a synergistic increase in total collagen production compared to TNF-α 1 ng/ml alone (Fig. 3A).Moreover, we found that inhibition of Akt (MK-2206), mTOR (Ridaforolimus) and ERK (PD98059), but not PI3K (Wortmannin), resulted in reduced total collagen production after stimulation with TMAO and TNF-α 1 ng/ml compared to DMSO treated cells after 96 h (Fig. 3B).Our findings suggest that the combination of TNF-α 1 ng/ml and TMAO synergistically increases total collagen production via Akt/mTOR and ERK pathways.

TNF-α and TMAO synergistically increase the release of cytokines from renal fibroblasts
Next, we continued to evaluate whether TMAO could enhance TNF-α mediated cytokine /cytokine receptor release from renal fibroblasts using a targeted protein analysis.We found that TMAO 300 μM and TNF-α alone increased the release of IL-6 compared to unstimulated cells after 24 h (Fig. 4).TNF-α was also found to increase LAP TGF-β-1 release compared to unstimulated cells (Fig. 4).We found that the combination of TNF-α 50 ng/ ml and TMAO significantly enhanced the release of LAP TGF-β-1, IL-6, SCF, LIF, CSF-1, IL-10RB and IL-18R1 compared to TNF-α 50 ng/ml alone (Fig. 4).LAP TGF-β-1 release was also significantly increased after TNF-α  www.nature.com/scientificreports/ 1 ng/ml and TMAO stimulation compared to TNF-α 1 ng/ml alone (Fig. 4).Taken together, our results suggest that the combination of TNF-α and TMAO can enhance the release of cytokines from renal fibroblasts.

TMAO and TNF-α synergistically increase the secretion of chemokines
In the next step we also wanted to evaluate whether TMAO could enhance TNF-α mediated chemokine release from renal fibroblasts.We found that TMAO 300 μM alone increased the release of IL-8 compared to unstimulated cells after 24 h (Fig. 5).TNF-α was found to increase IL-8, CXCL-1, CXCL-6, MCP-1, MCP-3 and CCL20 release compared to unstimulated cells (Fig. 5).In addition, we found that the combination of TNF-α 50 ng/ ml and TMAO significantly enhanced the release of MCP-1, MCP-3 and MCP-2 compared to TNF-α 50 ng/ml alone (Fig. 5).Moreover, the combination of TNF-α 10 ng/ml and TMAO, significantly increased the secretion of MCP-2 compared to TNF-α 10 ng/ml alone.The combination of TNF-α 1 ng/ml and TMAO also increased the release of CXCL-6, MCP-3 and CCL20 compared to TNF-α 1 ng/ml alone (Fig. 5).Hence, our results suggest that the combination of TNF-α and TMAO can enhance the release of several chemokines from renal fibroblasts.

Discussion
Several studies indicate that TMAO exacerbates tubulointerstitial fibrosis and renal inflammation in CKD 6,[21][22][23] .Furthermore, there is substantial evidence demonstrating that TNF-α plays a significant role in contributing to renal fibrosis 18,19 .However, the additive or synergistic contribution of TMAO and TNF-α on renal inflammation  We started by evaluating the effect of TMAO and TNF-α on fibronectin release from renal fibroblasts.We found that neither TMAO nor TNF-α increased fibronectin release from renal fibroblasts.This is in accordance with our previous findings 22 .However, we found that the combination of TMAO and TNF-α can increase fibronectin release compared to TNF-α alone.The observed fibronectin release was independent of fibroblast cell death.We have previously shown that TMAO is unable to induce increased fibronectin release from renal fibroblast up to 96 h posttreatment 22 .TMAO and TNF-α, independent of concentration, did not mediate increased fibroblast cell death.Fibronectin, a high molecular weight glycoprotein with adhesive properties, holds a pivotal function in both wound-healing processes and the formation of extracellular matrix 27 .Under pathophysiological conditions, fibronectin expression levels are dramatically increased in the renal tubulointerstitium which contributes to renal fibrosis.Taken together, our findings indicate that the combined exposure of TMAO and TNF-α can increase fibronectin release from renal fibroblasts.
We next evaluated the effect of TMAO and TNF-α on renal fibroblast proliferation and collagen production.We found that both TNF-α and TMAO induced increased fibroblast proliferation and that the combination of TNF-α 1 ng/ml and TMAO induced increased cell proliferation compared to TNF-α alone.However, this increase was more additive than synergistic.Furthermore, the overall difference between the treatment groups was very small, which strengthens the notion of an additive effect.We also found that TMAO and TNF-α alone or in combination increased total collagen production.The combination treatments of TNF-α 1 ng/ml and TMAO synergistically increased total collagen production compared to TNF-α alone.Next, we evaluated which signaling pathways TMAO and TNF-α activate to induce proliferation and collagen production.This was done to evaluate the mechanism behind the observed synergistic effects of TMAO and TNF-α.Our findings showed that both TMAO and TNF-α mediate their proliferative and collagen inducing effects on renal fibroblast via Akt, mTOR and ERK, but not PI3K.We have previously shown that TMAO induced increased renal fibroblast proliferation and collagen production via Akt and mTOR, but not via PI3K 22 .We have also shown that TMAO can reduce megalin expression in proximal tubular cells via PI3K and ERK 28 .In addition, TMAO has been linked to promote vascular inflammation via ERK activation 29 .TNF-α is also known to activate Akt, mTOR, and ERK in different cells 20,30,31 .Hence, the synergistic effects of TMAO and TNF-α could be explained by their ability to activate the same pathways.Taken together, our findings indicate that Akt, mTOR and ERK, but not PI3K, mediates the effect of TMAO and TNF-α on fibroblast proliferation and collagen production.
Our study is the first aiming to elucidate the combined effect of TMAO and TNF-α on renal fibroblasts through assessing aspects of renal fibrosis and inflammation.The current existing research has focused mainly on how TMAO or TNF-α alone affects the progress of kidney disease.However, the co-existence of TMAO and TNF-α (and/or other pro-inflammatory factors) in the renal interstitium is closer to the pathophysiologic background of kidney disease.
In conclusion, our findings showed that TMAO enhances TNF-α mediated renal fibroblast proliferation and collagen production via Akt/mTOR/ERK signaling pathway.We also observed that the combination of TMAO and TNF-α increased the release of several inflammatory mediators associated with kidney disease.To the best of our knowledge, this is the first study elucidating the synergistic effects of TMAO and TNF-α on renal inflammation and fibrosis.Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.